Trellis Bioscience has developed its patented CellSpot" technology into a robust platform for characterizing the secreted antibody from single human lymphocytes with regard to specificity and affinity against multiple antigens. For example, the platform was used to isolate a rare, high quality human antibody to Respiratory Syncytial Virus (RSV) that is now being prepared for human clinical testing by Trellis'licensee, MedImmune. In this project, we propose to use CellSpot to isolate therapeutic antibodies to combat rabies virus (RV) infections. Blood lymphocytes from adults who have been vaccinated against RV will be layered onto an antibody capture surface. After 5 hours of incubation, each cell's secreted antibody forms a "footprint" in the vicinity of the cell (~150 <m diameter). These footprints will then be incubated with fluorescent beads conjugated to the antigen target, which in this project will be the G protein of RV. The G protein is the same antigen used in rabies vaccines and has been shown to elicit protective antibodies. There are differences between G proteins of different strains, and there are both homologous, conserved regions and heterologous, variable regions on the G proteins of different RV strains. The most useful monoclonal antibodies are those which react against the broadest set of variant G proteins. Therefore, the antibody footprints will be screened with a panel of distinguishable fluorescent beads conjugated to the G-antigens from different strains. Spots that contain antibody to multiple G proteins will be detected under automated microscopic examination and the responsible cells will be isolated, followed by cDNA cloning of the antibody for further characterization following transfection into an expression system. These antibodies will be screened for physiological activity both by an in vitro neutralization assay and by an in vivo animal infection model to measure direct protection from infection. In Phase I of this project, we anticipate isolating ~15 human antibodies to the G protein, of which at least one will be shown to be protective in the animal model, confirming utility by a monoclonal comparable to that provided by immune gamma globulin, the current standard of care. Based on success in this Phase I objective, we will submit a Phase II application to advance the development of this antibody to clinical trial with a particular focus on reducing the cost of manufacturing to be competitive with immune gamma globulin. The overall objective of this project is a therapeutic antibody to RV which is well tolerated and highly effective in protecting against a broad range of RV strains. PUBLIC HEALTH RELEVANCE: This project will use an innovative microscopic cell analysis technology to screen human lymphocytes for cells producing antibodies to the rabies virus G protein. The antibodies selected will have the ability to protect animals and, ultimately humans, during rabies infection and will be developed as new therapeutic agents for this disease.